LDL Receptor mRNA Expression Research Articles

Analysis of low-density lipoprotein receptor gene mutations in a family with familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a common monogenic autosomal dominant disorder, primarily mainly caused by pathogenic mutations in the low-density lipoprotein receptor (LDLR) gene. Through phenotypic-genetic linkage analysis, two LDLR pathogenic mutations were identified in FH families: c.G1027A (p.Gly343Ser) and c.G1879A (p.Ala627Thr). Whole exome sequencing was conducted on the proband with familial hypercholesterolemia to identify the target gene and screen for potential pathogenic mutations. The suspicious responsible mutation sites in 14 family members were analyzed using Sanger sequencing to assess genotype-phenotype correlations. Mutant and wild type plasmids were constructed and transfected into HEK293T cells to evaluate LDLR mRNA and protein expression. In parallel, bioinformatics tools were employed to predict structural and functional changes in the mutant LDLR. Immunofluorescence analysis revealed no significant difference in the intracellular localization of the p.Gly343Ser mutation, whereas protein expression of the p.Ala627Thr mutation was decreased and predominantly localized in the cytoplasm. Western blotting has showed that protein expression levels of the mutant variants were markedly declined in both cell lysates and supernatants. Enzyme linked immunosorbent assay has demonstrated that LDLR protein levels in the supernatant of cell culture medium was not significant different from those of the wild-type group. However, LDLR protein levels in the cell lysate of both the Gly343Ser and Ala627Thr variants groups were significantly lower than those in the wild-type group. Bioinformatic predictions further suggested that these mutations may affect post-translational modifications of the protein, providing additional insight into the mechanisms underlying the observed reduction in protein expression. In this study, we identified two heterozygous pathogenic variants in the LDLR gene, c.G1027A (p.Gly343Ser) and c.G1879A (p.Ala627Thr), in a family with familial hypercholesterolemia. We also conducted preliminary investigations into the mechanisms by which these mutations contribute to disease pathology.

Variation of LDL cholesterol in response to the replacement of saturated with unsaturated fatty acids: a nonrandomized, sequential dietary intervention; the Reading, Imperial, Surrey, Saturated fat Cholesterol Intervention (“RISSCI”-1) study

BackgroundSerum low density lipoprotein (LDL) cholesterol shows marked interindividual variation in response to the replacement of saturated fatty acids (SFAs) with unsaturated fatty acids (UFAs). ObjectivesTo demonstrate the efficacy of United Kingdom guidelines for exchanging dietary SFAs for UFAs, to reduce serum LDL cholesterol and other cardiovascular disease (CVD) risk factors, and to identify determinants of the variability in LDL cholesterol response. MethodsHealthy males (n = 109, mean ± SD age 48 ± 11 y; BMI 25.1 ± 3.3 kg/m2), consumed a higher-SFA/lower-UFA diet for 4 wk, followed by an isoenergetic, lower-SFA/higher-UFA diet for 4 wk (achieved intakes SFA:UFA as % total energy 19.1:14.8 and 8.9:24.5, respectively). Serum LDL cholesterol, CVD risk markers, peripheral blood mononuclear cell (PBMC) gene expression, and dietary intakes were assessed at baseline and the end of each diet. ResultsTransition from a higher-SFA/lower-UFA to a lower-SFA/higher-UFA diet significantly reduced fasting blood lipids: LDL cholesterol (−0.50 mmol/L; 95% confidence interval [CI]: −0.58, −0.42), high-density lipoprotein (HDL) cholesterol (−0.11 mmol/L; 95% CI: −0.14, −0.08), and total cholesterol (TC) (−0.65 mmol/L; 95% CI:−0.75, −0.55). The dietary exchange also reduced apolipoprotein (apo)B, TC:HDL cholesterol ratio, non-HDL cholesterol, E-selectin (P < 0.0001), and LDL subfraction composition (cholesterol [LDL-I and LDL-II], apoB100 [LDL-I and LDL-II], and TAG [LDL-II]) (P < 0.01). There was also an increase in plasma biomarkers of cholesterol intestinal absorption (β-sitosterol, campesterol, cholestanol), and synthesis (desmosterol) (P < 0.0001) and fold change in PBMC LDL-receptor mRNA expression relative to the higher-SFA/lower-UFA diet (P = 0.035). Marked interindividual variation in the change in serum LDL cholesterol response (−1.39 to +0.77 mmol/L) to this dietary exchange was observed, with 33.7% of this variation explained by serum LDL cholesterol before the lower-SFA/higher-UFA diet and reduction in dietary SFA intake (adjusted R2 27% and 6.7%, respectively). APOE genotype was unrelated to serum LDL cholesterol response to SFA. ConclusionsThese findings support the efficacy of United Kingdom SFA dietary guidelines for the overall lowering of serum LDL cholesterol but showed marked variation in LDL cholesterol response. Further identification of the determinants of this variation will facilitate targeting and increasing the efficacy of these guidelines.The RISSCI-1 study was registered with ClinicalTrials.Gov (No. NCT03270527).

The expression of cholesterol synthesis and steroidogenic markers in females of the Chinese brown frog (Rana dybowskii) during pre-spawning and pre-hibernation.

The oviduct of the Chinese brown frog (Rana dybowskii) expands in pre-hibernation rather than in pre-spawning, which is one of the physiological phenomena that occur in preparation for hibernation. Steroid hormones are known to regulate oviductal development. Cholesterol synthesis and steroidogenesis may play important role in the expansion of the oviduct before hibernation. In this study, we investigated the expression patterns of the markers that are involved in the de novo steroid synthesis pathway in the oviduct of R. dybowskii during pre-spawning and pre-hibernation. According to histological analysis, the oviduct of R. dybowskii contains epithelial cells, glandular cells, and tubule lumens. During pre-hibernation, oviductal pipe diameter and weight were significantly larger than during pre-spawning. 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR), Low-density lipoprotein receptor (LDLR), steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and steroidogenic factor 1 (SF-1) were detected in epithelial cells in pre-hibernation and glandular cells during pre-spawning. HMGCR, LDLR, StAR, and P450scc protein expression levels were higher in pre-hibernation than during pre-spawning, but the SF-1 protein expression level did not significantly differ. HMGCR, LDLR, StAR, P450scc (CYP11A1), and SF-1 (NR5A1) mRNA expression levels were significantly higher in pre-hibernation compared to pre-spawning. The transcriptome results showed that the steroid synthesis pathway was highly expressed during pre-hibernation. Existing results indicate that the oviduct is able to synthesize steroid hormones using cholesterol, and that steroid hormones may affect the oviductal functions of R. dybowskii.

Dihydromyricetin promotes LDL metabolism in HepG2 cells through the PCSK9/LDLR pathway

ABSTRACT Low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) play a pivotal role by regulating plasma low-density lipoprotein cholesterol (LDL-c) levels. Dihydromyricetin (DMY), the most abundant natural flavonoid in rattan tea, has proven anti-atherogenic effects, but the underlying molecular mechanisms remain poorly understood. Therefore, we studied the effects of DMY on LDLR and PCSK9. The results showed DMY promoted LDLR protein and mRNA expression and increased LDL uptake in HepG2 cells. DMY inhibited intracellular PCSK9 protein and mRNA expression. And it also significantly reduced PCSK9 levels in the cell culture medium. Furthermore, DMY inhibited the expression of PCSK9 through the liver nuclear transcription factor 1 A (HNF1A) and increased the protein expression of LDLR. Taken together, our results support the idea that DMY regulates LDL-c metabolism through PCSK9/LDLR signaling. This study reveals the potential mechanism of DMY’s anti-atherogenic effect and provides a theoretical basis for dietary DMY supplementation.

Lactobacillus reuteri NCIMB 30242 (LRC) Inhibits Cholesterol Synthesis and Stimulates Cholesterol Excretion in Animal and Cell Models.

In this study, we evaluated the effects of Lactobacillus reuteri NCIMB (LRC™) supplementation on hypercholesterolemia by researching its effects on cellular cholesterol metabolism in hypercholesterolemic rats (KHGASP-22-170) and HepG2 cell line. Rats were separated into six groups after adaptation and were then fed a normal control (NC), a high-cholesterol diet (HC), or a HC supplemented with simvastatin 15 mg/kg body weight (positive control [PC]), LRC 1 × 109 colony-forming units (CFU)/rat/day, LRC 4 × 109 CFU/rat/day, or LRC 1 × 1010 CFU/rat/day (1 × 109, 4 × 109, or 1 × 1010). The rats were dissected to study the effects of LRC on cholesterol metabolism and intestinal excretion at the end of experimental period. We discovered that LRC mainly participated in the restraint of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the uptake of low-density lipoprotein (LDL) cholesterol into tissues, partially in the transport of cholesteryl esters into high density lipoprotein for maturation, and intestinal excretion of cholesterol. These results are supported by the expression of transcription factors and enzymes such as HMG-CoA reductase, SREBP2, CYP7A1, CETP, and LCAT in both messenger RNA (mRNA) and protein levels in serum and hepatic tissue. Furthermore, the LRC treatment in HepG2 significantly reduced the mRNA expression of HMG-CoA reductase, SREBP2, and CEPT and significantly increased the mRNA expression of LDL-receptor, LCAT, and CYP7A1 at all doses. Hence, we suggest that LRC supplementation could alleviate the serum cholesterol level by inhibiting the intracellular cholesterol synthesis, and augmenting excretion of intestinal cholesterol.

Caffeine can alleviate non-alcoholic fatty liver disease by augmenting LDLR expression via targeting EGFR.

Increasing low-density lipoprotein receptor (LDLR) protein levels represents a key strategy for the prevention and treatment. Berberine can reportedly alleviate non-alcoholic fatty liver disease (NAFLD) by increasing the LDLR expression in an ERK1/2 signaling-dependent manner of NAFLD. Studies have shown that caffeine can inhibit fat deposition in the livers of mice; however, caffeine has not been reported to alleviate NAFLD by augmenting the LDLR expression via targeting EGFR. Here, an MTT assay, western blotting, RT-qPCR, immunohistochemistry, and surface plasmon resonance (SPR) analysis were used to investigate the role of caffeine in low-density lipoprotein cholesterol (LDL-C) clearance both in vitro and in vivo. In vitro, we found that caffeine could activate the EGFR-ERK1/2 signaling pathway in HepG2 cells, leading to increased LDLR mRNA and protein expression, and this effect could be inhibited by cetuximab. The SPR assay results have indicated that caffeine may increase the LDLR expression by directly binding to the EGFR extracellular domain and activating the EGFR-ERK1/2 signaling pathway. In vivo, caffeine markedly improved fatty liver and related blood indices in ApoE KO mice with high-fat-diet-induced NAFLD. Consistent with our in vitro results, we found that caffeine could also activate EGFR-ERK1/2 signaling and promote the LDLR expression in ApoE KO mice. In summary, caffeine can enhance the LDLR expression by directly binding to EGFR and activating the EGFR-ERK1/2 signaling pathway. EGFR signaling may represent a novel target for the prevention and treatment of NAFLD.

Pterostilbene Increases LDL Metabolism in HL-1 Cardiomyocytes by Modulating the PCSK9/HNF1α/SREBP2/LDLR Signaling Cascade, Upregulating Epigenetic hsa-miR-335 and hsa-miR-6825, and LDL Receptor Expression.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) can promote the degradation of low-density lipoprotein (LDL) receptor (LDLR), leading to hypercholesterolemia and myocardial dysfunction. The intracellular regulatory mechanism by which the natural polyphenol pterostilbene modulates the PCSK9/LDLR signaling pathway in cardiomyocytes has not been evaluated. We conducted Western blotting, flow cytometry, immunofluorescence staining, and mean fluorescence intensity analyses of pterostilbene-treated mouse HL-1 cardiomyocytes. Pterostilbene did not alter cardiomyocyte viability. Compared to the control group, treatment with both 2.5 and 5 μM pterostilbene significantly increased the LDLR protein expression accompanied by increased uptake of LDL. The expression of the mature PCSK9 was significantly suppressed at the protein and mRNA level by the treatment with both 2.5 and 5 μM pterostilbene, respectively, compared to the control. Furthermore, 2.5 and 5 μM pterostilbene treatment resulted in a significant reduction in the protein hepatic nuclear factor 1α (HNF1α)/histone deacetylase 2 (HDAC2) ratio and sterol regulatory element-binding protein-2 (SREBP2)/HDAC2 ratio. The expression of both hypoxia-inducible factor-1 α (HIF1α) and nuclear factor erythroid 2-related factor 2 (Nrf2) at the protein level was also suppressed. Pterostilbene as compared to short hairpin RNA against SREBP2 induced a higher protein expression of LDLR and lower nuclear accumulation of HNF1α and SREBP2. In addition, pterostilbene reduced PCSK9/SREBP2 interaction and mRNA expression by increasing the expression of hsa-miR-335 and hsa-miR-6825, which, in turn, increased LDLR mRNA expression. In cardiomyocytes, pterostilbene dose-dependently decreases and increases the protein and mRNA expression of PCSK9 and LDLR, respectively, by suppressing four transcription factors, HNF1α, SREBP2, HIF1α, and Nrf2, and enhancing the expression of hsa-miR-335 and hsa-miR-6825, which suppress PCSK9/SREBP2 interaction.

Identification of neolignans with PCSK9 downregulatory and LDLR upregulatory activities from Penthorum chinense and the potential in cholesterol uptake by transcriptional regulation of LDLR via SREBP2

Ethnopharmacological relevancePenthorum chinense has been used in East Asia for the treatment of cholecystitis, infectious hepatitis, jaundice and to treat liver problems. Recent evidences provided the potential for the clinical use of P. chinense in the treatment of metabolic disease.Aim of the study: Based on the traditional use and recent evidences, we investigated the effects of constituents from P. chinense with modulation on proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR) expression, and the effect of the most active substance on cholesterol uptake, and genes relevant to lipid metabolism. Materials and methodsThe isolation of compounds from the BuOH-soluble extract of 80% methanol extract of P. chinense was conducted using chromatographic methods and the structures were established by interpreting spectroscopic data. Quantitative real time-PCR, and Western blot analysis were performed to monitor the regulatory activity on PCSK9 and LDLR expression. PCSK9-LDLR binding interaction was also tested. The cholesterol uptake in hepatocyte was measured using 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI)-labeled LDL cholesterol. Additionally, gene network analysis of LDLR and responses of its target proteins were carried out to discover genes germane to the effect of active compound on HepG2 cells. Moreover, we performed protein-protein interaction analysis via String and constructed the compound target network using Cytoscape. ResultsTwo new neolignans and 37 known compounds were characterized from P. chinense. Of the isolated compounds, (7′E,8S)-2′,4,8-trihydroxy-3-methoxy-2,4′-epoxy-8,5′-neolign-7′-en-7-one (3), penthorin A (4) and methyl gallate (25) were found to suppress PCSK9 mRNA expression with IC50 values of 5.13, 15.56 and 11.66 μM, respectively. However, all the isolated compounds were found to be inactive in PCSK9-LDLR interaction assay. Additionally, a dibenzoxepine-type lignan analog, (7′E,8S)-2′,4,8-trihydroxy-3-methoxy-2,4′-epoxy-8,5′-neolign-7′-en-7-one (3) demonstrated to upregulate LDLR mRNA and protein expression via transcriptional factor sterol regulatory element-binding protein 2 (SREBP2). Furthermore, (7′E,8S)-2′,4,8-trihydroxy-3-methoxy-2,4′-epoxy-8,5′-neolign-7′-en-7-one (3) increase the LDL-cholesterol uptake in DiI-LDL assay. Conclusion(7′E,8S)-2′,4,8-trihydroxy-3-methoxy-2,4′-epoxy-8,5′-neolign-7′-en-7-one (3) seemed to increase potentially cholesterol uptake via the downregulation of PCSK9 and the activation of LDLR in hepatocytes. Moreover, SREBP2 was found to play an important role in regulation of PCSK9 and LDLR by (7′E,8S)-2′,4,8-trihydroxy-3-methoxy-2,4′-epoxy-8,5′-neolign-7′-en-7-one.

HSP25 Vaccination Attenuates Atherogenesis via Upregulation of LDLR Expression, Lowering of PCSK9 Levels and Curbing of Inflammation.

[Figure: see text].

Amplification of the Hazelnut-Induced Epigenetic Modulation of LDLR Gene Expression in THLE-2 Human Primary Hepatocytes Compared to HepG2 Hepatocarcinoma Cells

Dietary supplementation with tree nuts, including hazelnuts, has been associated with reduced cardiovascular disease risk factors, due to improved blood lipid profile. In the attempt to identify the molecular mechanism(s) underlying such beneficial effect, we here characterized the response of the human primary hepatocytes (THLE-2 cells) to the administration of an ethanolic extract of hazelnut (Corylus avellana L., cultivar Tonda Gentile Romana) in terms of regulation of the Low-Density Lipoprotein Receptor (LDLR), a major blood cholesterol carrier that positively correlates with improved blood lipids level. We demonstrated that hazelnut (0.004-0.4 mg/ml) does not alter viability and growth of primary liver cells, but significantly stimulates (P&lt;0.05) the LDLR mRNA and membrane protein expression. We also proved that LDLR increase is driven by an epigenetic-based mechanism, as hazelnut treatments trigger a strong DNA hypo-methylation of two CpG-rich regions in the LDLR gene promoter, mapping at -739/-548 and -221/+174 position. The hazelnutmediated hypo-methylation is much stronger and extensive in THLE-2 than in HepG2 human hepatocarcinoma cells, supporting the significance of the liver context for performing studies on dietary supplementation. As overall, our findings demonstrate that hazelnut triggers an epigenetic-dependent regulation of LDLR expression in human primary liver cells in vitro, thus identifying in the LDLR stimulation a molecular mechanism contributing to the health promoting functions of hazelnuts.

Cigarette smoke extract stimulates PCSK9 production in HepG2 cells via ROS/NF‑κB signaling.

Cigarette smoke (CS) exposure is a risk factor for dyslipidemia and atherosclerosis. Reduced expression of low-density lipoprotein receptor (LDLR) in hepatocytes may be one of the underlying mechanisms for these disorders. The aim of the present study was to investigate the molecular mechanism underlying the regulatory effect of CS extract (CSE) on proprotein convertase subtilisin/kexin type 9 (PCSK9) and low LDLR expression in HepG2 cells. PCSK9 and LDLR mRNA and protein expression levels in HepG2 cells were evaluated after CSE treatment via reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. In addition, total intracellular reactive oxygen species (ROS) production was determined via 2,7-dichlorofluorescein diacetate fluorescence. CSE significantly increased PCSK9 expression and inhibited LDLR expression in a time- and concentration-dependent manner. Furthermore, CSE significantly induced ROS production and nuclear factor κB (NF-κB) activation. However, pretreatment with a ROS scavenger or an NF-κB inhibitor significantly attenuated the CSE-induced changes in PCSK9 and LDLR expression. In addition, pretreatment with melatonin markedly reduced ROS production, NF-κB activation and PCSK9 expression, and increased LDLR expression in the CSE-treated cells. These data suggest that melatonin inhibits CSE-regulated PCSK9 and LDLR production in HepG2 cells via ROS/NF-κB signaling.

Deficiency of Nardilysin in the Liver Reduces Serum Cholesterol Levels.

Nardilysin (NRDC) has been shown to be involved in post-translational histone modifications, in addition to enhancement in ectodomain shedding of membrane-anchored protein, which play significant roles in various pathophysiology, including glucose homeostasis, inflammatory diseases and cancer. The present study sought to determine roles of NRDC in the liver on lipid and lipoprotein metabolism. We established liver-specific NRDC deficient mice by use of NRD1 floxed mice and albumin promoter-Cre recombinase (Cre) transgenic mice, and found that their serum low-density lipoprotein (LDL) cholesterol levels were significantly lower than those in control littermate mice. In the liver, LDL receptor (LDLR) mRNA expression was significantly upregulated, while inducible degrader of LDLR (IDOL) and microsomal triglyceride transfer protein (MTP) mRNA expression was significantly downregulated, in liver-specific NRDC deficient mice. Hepatic cell-surface LDLR expression levels were significantly elevated and serum pro-protein convertase subtilisin-kexin type 9 (PCSK9) levels were significantly reduced in mice with hepatic NRDC deficiency. In cultured hepatocytes, NRDC deficiency significantly reduced secreted PCSK9 and increased cell-surface LDLR expression. On the other hand, NRDC overexpression in cultured hepatocytes significantly increased secreted PCSK9 and lowered cell-surface LDLR expression. Thus, NRDC in murine hepatocytes appears to play key roles in cholesterol homeostasis, although the precise molecular mechanisms remain to be determined.

Role of Genetic & Epigenetic Modifications of Low-Density Lipoprotein Receptor (LDLR) Gene in South Indian Acute Myocardial Infraction Patients

Background: Acute Myocardial Infarction (AMI) has conventional risk factors as well as genetic and epigenetic modifications of various genes. Among them gene coding for Low density lipoprotein receptor (LDLR) plays a strategic role in cholesterol homeostasis by eliminating excess LDL-C in blood. LDLR gene promoter polymorphism & DNA methylation status might regulate the expression of genes & its activity. Objective: The present study is designed to evaluate the association of LDLR gene promoter c.-139delCTCCCCCTGC polymorphism & DNA methylation status with Acute Myocardial Infarction (AMI). Methods: 300 angiographically documented MI patients and 300 healthy individuals were recruited for the study. Genomic DNA & RNA was extracted from the whole blood using commercially available assay kits further RNA was converted to cDNA. Biochemical parameters like Total cholesterol, LDL-C, HDL-C, and Triglycerides were estimated using kit methods. Results: The risk factors like systolic, diastolic blood pressures, hypertension, diabetes mellitus, smoking, lipids were found to be significantly higher in AMI patients compared to controls. The genotype and allele distributions of the LDLR c.-139del polymorphism revealed that DD genotype & D-allele were found to be higher among the AMI patients than that of controls. Furthermore, we have observed LDLR gene differential promoter DNA methylation status & mRNA expression. In addition, Insilco analysis have shown that there is a change in TF binding at the Insertion & Deletion sites. Conclusion: The findings of the study suggest that the LDLR c.-139del DD genotype might be used as prognostic marker in risk prediction of Acute Myocardial Infarction.

Dyslipidemia in Chinese Pancreatic Cancer Patients: A Two-Center Retrospective Study.

Background: Pancreatic cancer (PC) is one of the most aggressive and lethal malignancies in the world. High cholesterol intake may have a certain association with an elevated risk of PC, though dyslipidemia in PC patients has rarely been reported. In this study, we compared serum lipids levels between PC and non-PC tumor patients and assessed their prognostic value in PC.Methods: 271 patients treated at Wuhan Union Hospital from January 2012 to December 2016 and 204 individuals at Shanghai General Hospital from January 2018 to December 2019 were recruited. Their demographic parameters, laboratory data, pathological information, and clinical outcomes were extracted and analyzed. The mRNA expressions of related lipoprotein, low density lipoprotein receptor (LDLR) and high density lipoprotein binding protein (HDLBP), in PC tissues and paired noncancerous tissues and follow-up information were assessed based on the GEO database (GSE15471 and GSE62165) and TCGA database.Results: A total of 172 non-PC tumor patients and 260 PC patients were finally eligible for our analysis. PC patients exhibited higher levels of serum triglyceride, cholesterol, and low-density lipoprotein (LDL) and a lower serum high-density lipoprotein (HDL) level on admission versus the non-PC tumor group. In PC patients, LDLR mRNA expression was upregulated, and HDLBP mRNA expression was downregulated in cancerous tissues compared to these levels in paired noncancerous tissues. The survival analysis revealed that dyslipidemia had a non-significant association with a poor prognosis, but PC patients with a high LDLR level were at risk of poor survival.Conclusion: Dyslipidemia is detected in PC patients but has a non-significant relation to PC prognosis. However, LDLR may be a potential predictive marker for PC prognosis.

Oral administration of trehangelin-A alleviates metabolic disorders caused by a high-fat diet through improvement of lipid metabolism and restored beneficial microbiota

The present study investigated whether or not the oral administration of trehangelin-A (THG-A) is effective for metabolic disorders caused by a high-fat diet, as we previously showed that the intraperitoneal administration of THG-A improved metabolic disorders caused by a high-fat diet. Mice received a control diet or high-fat diet for eight weeks. Concurrently, mice were orally administered 0.2 ml/mouse phosphate-buffered saline (PBS) or 1 or 10 mg/0.2 ml/mouse of THG-A once daily during the experiment. The weight gain caused by a high-fat diet was significantly suppressed by oral THG-A compared to a high-fat diet without THG-A. In addition, at eight weeks after starting the diet, the increased plasma total-cholesterol (T-CHO) and low-density lipoprotein-cholesterol (LDL-C) levels caused by a high-fat diet were significantly reduced by 10 mg/mouse THG-A and tended to attenuated by 1 mg/mouse THG-A. The LDL receptor and CYP7A1 mRNA expression in liver associated with lipid metabolism for reducing plasma LDL-C levels was significantly enhanced by oral THG-A. In contrast, oral THG-A exerted no marked effects on mice fed the control diet. The dysbiosis of a high-fat diet fed mice, which is in the form of an increased Firmicutes-to-Bacteroidetes ratio, also recovered, and the high-fat diet induced decreased levels of Bacteroides and Akkermansia genera, which are beneficial microbiota against metabolic disorders, were also restored by oral THG-A. These results indicate that oral THG-A administration acts on metabolic disorders by improving the lipid metabolism and restoring beneficial microbiota to resolve high-fat diet induced dysbiosis.

SAT-151 Regulation of Low-Density Lipoprotein Receptor Expression in Triple Negative Breast Cancer

Preclinical models and clinical studies suggest that hypercholesterolemia promotes breast cancer progression 1,2. The expression of the low-density lipoprotein receptor (LDLR) has been positively associated with poorer recurrence-free survival in human breast cancer studies 3. Mechanistically, LDLR has been demonstrated to play a role in the increased tumor growth associated with hypercholesterolemia, as knock-down of LDLR led to decreased tumor growth in setting of elevated circulating LDL cholesterol. The aim of this study was to identify factors which up-regulate expression of LDLR in triple negative breast cancer (TNBC). In glioblastoma, hyper-activation of the epidermal growth factor receptor (EGFR) signaling pathway has been associated with greater LDLR expression and susceptibility to targeting of cholesterol metabolism4. As EGFR is frequently expressed in TNBC5, we examined if increased LDLR expression is associated with activation of the EGFR signaling pathway in TNBC. The expression of LDLR in the TNBC cell lines, MDA-MB-231 (231) and MDA-MB-468 (468) was examined pre- and post-EGF stimulation of the EGFR and in the presence of chemical inhibitors. Cells were grown in DMEM/10% FBS/1% Pen/strep (P/S), and experiments were performed under reduced serum conditions at 1.25%FBS/DMEM/1%P/S. In the absence of stimulation, LDLR protein expression was 3-fold higher in 231 vs 468 cell lines. This was despite mRNA expression being comparable at baseline, suggesting that the difference in protein expression was post-transcriptionally mediated. Treatment with 10 ng/mL EGF for 2 hours led to an increased activation of the EGFR, phosphorylation of Akt and extracellular signal regulated kinase (ERK) in both cell lines but induced an increase in LDLR protein and mRNA expression only in 468 cells. Treatment of 468 cells with EGF after exposure to actinomycin, a transcription inhibitor, revealed that EGF treatment resulted in reduced degradation of LDLR mRNA (p = 0.002) over 3 hours, suggesting that the EGF-induced increase in LDLR expression was by protection of LDLR mRNA from degradation. Chemical inhibition of the ERK pathway with 20 μM UO126 reduced both the EGF-induced increase in LDLR expression in 468 cells (p = 0.015) as well as the high baseline expression of LDLR by half in 231 cells (p = 0.001). Overall our results suggest that the EGFR/ERK signaling pathway regulates LDLR expression in TNBC, supporting the increased anabolic needs of this aggressive, swiftly expanding form of breast cancer.

Inhibitors of AKT kinase increase LDL receptor mRNA expression by two different mechanisms.

Protein kinase B (AKT) is a serine/threonine kinase that functions as an important downstream effector of phosphoinositide 3-kinase. We have recently shown that MK-2206 and triciribine, two highly selective AKT inhibitors increase the level of low density lipoprotein receptor (LDLR) mRNA which leads to increased amount of cell-surface LDLRs. However, whereas MK-2206 induces transcription of the LDLR gene, triciribine stabilizes LDLR mRNA, raising the possibility that the two inhibitors may actually affect other kinases than AKT. In this study, we aimed to ascertain the role of AKT in regulation of LDLR mRNA expression by examining the effect of five additional AKT inhibitors on LDLR mRNA levels. Here we show that in cultured HepG2 cells, AKT inhibitors ARQ-092, AKT inhibitor VIII, perifosine, AT7867 and CCT128930 increase LDLR mRNA levels by inducing the activity of LDLR promoter. CCT128930 also increased the stability of LDLR mRNA. To study the role of AKT isoforms on LDLR mRNA levels, we examined the effect of siRNA-mediated knockdown of AKT1 or AKT2 on LDLR promoter activity and LDLR mRNA stability. Whereas knockdown of either AKT1 or AKT2 led to upregulation of LDLR promoter activity, only knockdown of AKT2 had a stabilizing effect on LDLR mRNA. Taken together, these results provide strong evidence for involvement of AKT in regulation of LDLR mRNA expression, and point towards the AKT isoform specificity for upregulation of LDLR mRNA expression.

Enhancement of the antioxidant and hypolipidemic activities of Puerariae radix by fermentation with Aspergillus niger.

Puerariae radix (PR) is a traditional Chinese food and medicine. In this study, the chemical profile and bioactivities of PR fermented with Aspergillus niger (PFA) were investigated. Based on HPLC analysis, PFA chemical profile changed and total phenols increased by 12.5% relative to PR. Consequently, the in vitro antioxidant activity significantly improved. According to the blood lipid analysis in mice, PFA showed a better ability to inhibit the increased blood lipid levels induced by Triton WR-1339 than PR. In a quantitative real-time PCR (qRT-PCR) study, PFA up-regulated cholesterol 7-alpha hydroxylase (CYP7A1) and low-density lipoprotein receptor mRNA expression, which were 50% and 44.8% higher than the levels induced by high-dose PR. Conversely, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR) mRNA expression was down-regulated to 46.8% lower than the levels induced by high-dose PR. The qRT-PCR results suggested that PFA displayed better hypolipidemic activity than PR, due to its superior ability to regulate mRNA expression.

Hepatoprotective effects of Cassia semen ethanol extract on non-alcoholic fatty liver disease in experimental rat

Context: Cassia semen (Cs), a seed of Cassia obtusifolia L. (Leguminosae), is a popular functional beverage. Previous studies reported that Cs displayed antioxidant, antifungal and strong liver protective effects.Objective: This study evaluates the hepatoprotective effects of Cs on non-alcoholic fatty liver disease (NAFLD).Materials and methods: Seventy-two male Wistar rats raised with high-fat diet (HFD) were randomly allotted into model, metformin (0.2 g/kg) and Cs (0.5, 1, and 2 g/kg)-treated groups. Another 12 rats were raised with normal feed as control group; all the rats were orally administrated with drugs and vehicle for 6 weeks. Alanine transferase (ALT), aspartate transaminase (AST), triglycerides (TG), total cholesterol (TC), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8 and low density lipoprotein receptor (LDL-R) mRNA levels were measured at the end of the experiment.Results: Twelve weeks of HFD administration significantly increased the levels of AST, ALT, TG, TC, TNF-α, IL-6, IL-8 and MDA, decreased SOD (199.42 vs. 137.70 U/mg protein) and GSH (9.76 vs. 4.55 mg/g protein) contents, compared to control group. Cs administration group significantly decreased the elevated biomarkers with the ED50 = 1.2 g/kg for NAFLD rats. Cs treatment also prevents the decreased expression of LDL-R mRNA, and improved the histopathological changes compared to model group.Conclusions: The hepatoprotective effect of Cs on NAFLD may possibly be due to its antioxidant effect. Cs may become a potent hepatoprotective agent in clinical therapy in the future.

Protective effect of atorvastatin meditated by HMGCR gene on diabetic rats with atherosclerosis: An in vivo and in vitro study

BackgroundAccelerated atherosclerosis in patients suffering from diabetes represents a major cause of morbidity and mortality. The aim of present study was to investigate the protective effects conferred by atorvastatin (AVT) meditated by the HMGCR gene in diabetic rats with atherosclerosis. MethodsSerum triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), very-low-density lipoprotein cholesterol (VLDL-C), fasting blood glucose (FBG) and serum insulin (INS) were all determined by means of in vivo experiments. Following the establishment of the diabetic model of atherosclerosis, the expressions of HMGCR, low density lipoprotein receptor (LDLR), fatty acid synthase (FASN) were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis in the vitro experiments. Flow cytometry was adopted in order to detect cell cycle and apoptosis. ResultsThe in vivo experiments results indicated that FBG and INS among the diabetic arteriosclerosis rats exhibited markedly higher levels; after injected with AVT and HMGCR, decreased contents of TC, TG, LDL-C and VLDL-C, while increased contents of HDL-C as well as an increased positive rate of HMGCR protein expression were observed. In vitro experiment, the mRNA and protein expression of LDLR were increased and FASN were decreased in cells transfected with HMGCR and AVT; with a greater number of cells arrested at the S phase and less in the G0/G1 phase, as well as data indicating the rate of apoptosis was inhibited after HMGCR and AVT transfection processes. ConclusionThe key findings of the present study suggested that the protective effect conferred by AVT in diabetic rats with atherosclerosis was associated with the overexpression of the HMGCR gene, thus presenting a novel target for atherosclerosis treatment.